Putting genome analysis to good use: Lessons from C-reactive protein and cardiovascular disease
ABSTRACTNew methods of studying the human genome offer novel ways to examine the relationship between biomarkers and common, chronic human diseases. As an example, we will review a large genomics study (Elliott et al, JAMA 2009; 302:37–48) that concluded that C-reactive protein (CRP) is likely not a cause of coronary heart disease, although it is a marker for it.
KEY POINTS
- Genome-wide association studies can uncover associations between genetic markers and medical conditions, but they fall short of establishing causality or even clear biologic interactions between a genetic variant and a disease state.
- Mendelian randomization is a method for addressing the relationship between genetic variants and disease, ie, whether a biomarker affected by the variant is a cause of the disease or merely a bystander.
- CRP, an acute-phase reactant produced by the liver in response to inflammation, is one of many inflammatory markers whose levels correlate with coronary disease and which has been suggested to play a role in its pathogenesis.
- The findings of Elliott et al suggest that therapies that specifically lower CRP levels are not likely to affect coronary artery disease.
C-reactive protein: Marker or mediator?
Unlike LDL-C, no familial syndromes of coronary heart disease have been recognized in patients who have isolated high serum levels of CRP.
Since many substances in addition to CRP increase in concentration in both acute and chronic inflammatory states, agents that lower CRP in a targeted manner would be needed for large prospective, randomized trials to show whether CRP plays a direct role in coronary heart disease. A specific CRP inhibitor, 1,6-bis(phosphocholine)-hexane, may aid in these efforts, although it is not orally bioavailable and has a very short serum half-life.24
The JUPITER trial. Statins lower levels of both LDL-C and CRP. The Justification for the Use of Statins in Primary Prevention: an Intervention Evaluating Rosuvastatin (JUPITER) trial was designed to find out whether statins alter coronary risk in patients with “normal” LDL-C levels (< 130 mg/dL) and elevated CRP levels (> 2 g/L).25
In this prospective, randomized trial, statin treatment resulted in a dramatic risk reduction of 40% to 50% in multiple coronary end points, as well as a reduction in CRP levels of 37% compared with placebo. However, LDL-C levels fell by 50%, confounding the effect on CRP, as the lower coronary event rate could alternatively be explained by the effect of lower-than-normal LDL-C levels. Thus, a causative link between CRP and coronary heart disease could not be proved.26
Though ongoing trials may further illuminate the role of inflammation in the development of coronary heart disease, and specific CRP inhibitors are in development, we have few tools to answer the fundamental question of whether CRP itself is an active participant in cardiovascular disease progression or if it is a bystander marker, helping to define risk for patients who develop coronary heart disease without other known risk factors.
Of note, adding CRP to the Framingham risk score does not improve its predictive power very much in any age group.27,28 Nevertheless, for certain end points, such as the long-term rate of death after percutaneous coronary intervention29 or of cardiovascular death immediately after coronary artery bypass grafting,30 CRP levels predict coronary events reliably.
BIOMARKERS AND MENDELIAN RANDOMIZATION
Further insight into the CRP-coronary association may lie in the genes. Intriguingly, while mutations have been found that alter the serum concentration of CRP, these isolated changes in CRP levels have not yet been shown to affect heart disease risk.9,31,32
If one were to design a prospective, interventional study to evaluate the role of CRP in coronary heart disease, it would be very difficult to tease apart the specific impact of CRP from that of other variables that are often present in people with high CRP, such as obesity and hyperlipidemia. The technique of mendelian randomization offers a way to evaluate the correlation between coronary heart disease development and CRP levels independent of other risk factors.
How many heart attacks in people with or without polymorphisms?
Mendelian randomization takes advantage of a basic genetic principle, ie, the independent assortment of traits. According to Mendel’s second law, alleles for different traits are inherited independently of one another. Therefore, the gene that encodes CRP and other genes that influence its circulating level are presumably inherited independently from other genes that influence coronary risk.
In typical studies of CRP, participants are grouped according to whether they have high or low CRP levels. In these studies, confounding variables congregate in these two groups. For example, people with high CRP may be more likely to smoke and to have a higher body mass index and higher lipid levels—all of which influence cardiovascular outcomes. It is therefore difficult to tease out the effect of CRP levels from other background risk factors.
In contrast, in studies using mendelian randomization, patients are grouped according to whether they have a variant that affects the substance being studied (eg, CRP), and outcomes are compared between the two genetic groups.
Strengths and limitations of this method
By randomizing research subjects by gene variants affecting CRP levels, it is theoretically possible to achieve more equal stratification and minimize confounding between subgroups.33
Mendelian randomization should also address the possibility of “reverse causality,” when the intermediate trait with a potential role in disease development (eg, CRP) is actually regulated by the disease state itself (ie, “inflammation of atherosclerotic cardiovascular disease”).34
A limitation of mendelian randomization is that different genes influencing the biomarker under investigation must be proven to be truly randomly assorted among populations. It cannot be assumed that levels of a biomarker are equally distributed across cases and controls when there may in fact be non-random genetic associations.
For instance, if SNPs in various genes that affect creatine kinase levels were being compared to cardiovascular outcome, it would be important to take into account that baseline creatine kinase levels are higher in African Americans as well as in men in interpreting the study data.35
THE ELLIOTT STUDY (2009)
In a study published in 2009, Elliott et al1 mined genome-wide data collected over the last decade to bring more clarity to the issue of causality between elevated CRP and heart disease.
To accomplish mendelian randomization, the authors assessed SNPs that affect circulating CRP levels in combined sets of 28,000 cases and 100,000 controls—robust population sizes. The SNP variants included were associated with approximately 20% lower CRP levels. This degree of CRP reduction should correspond to a 6% reduction in coronary risk as predicted by meta-analysis of observational studies.
